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Application of SYBRgreen PCR and 2DGE methods to authenticate edible bird's nest food
Food Research International 43 (2010) 2020–2026
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Food Research International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s
Application of SYBRgreen PCR and 2DGE methods to authenticate edible bird's nest food Yajun Wu a, Ying Chen a,⁎, Bin Wang a, Liqun Bai a, Wu ri han a, Yiqiang Ge b, Fei Yuan a a b
Chinese Academy of Inspection and Quarantine, No.3, Gaobeidian North Street, Chaoyang District, Beijing, China College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China
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Article history: Received 31 March 2010 Accepted 30 May 2010 Keywords: Edible bird's nest Authentication SYBRgreen PCR 2DGE
a b s t r a c t Edible bird's nest (EBN) as a special kind of food tonic has been highly esteemed in Chinese cuisine and medicinal culture. Particularly with the discovery of its healthy function by modern science, consumption of EBN food gained greater popularity within Chinese community and outside. Authentication of this precious and expensive food material became an urgent task facing the increasing occurrence of adulteration in the market. Herein we reported the combination of DNA based PCR and protein based two dimensional gel electrophoresis (2DGE) methods for rapid and reliable identification of genuine EBN product. Fourteen EBN samples from different countries were studied. PCR method was proved to be able to differentiate EBN and the other biological materials and it could detect EBN ingredient from 0.5% EBN/Tremella fungus mixture. 2DGE method was proved to be feasible and versatile in EBN identification because of the simple and unique protein pattern of EBN. The method could detect 10% Tremella fungus from EBN. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction EBN originated from the saliva of four swiftlet species: Collocalia fuciphaga, C. germanis, C. maxima and C. unicolor (Goh et al., 2001). Due to the highly evaluated function both nutritiously (water-soluble protein, carbohydrate, iron, inorganic salt and fiber) and medically (anti-aging, anti-cancer, immunity-enhancing, etc), EBN has been esteemed a precious food tonic by Chinese people ever since the Tang dynasty (618 AD). It was even referred as “Caviar of the East”. Current scientific study confirmed that EBN has haemagglutination inhibiting activity against the influenza virus and contained avian epidermal growth factor (Marcone, 2005; Lin et al., 2006). Nowadays with the help of modern commercialization and technology, EBN was developed into various food products including drink, food additive, and even cosmetic ingredient. At present EBN raw material mainly comes from Asian countries, such as China, Malaysia, Indonesia, Vietnam, Thailand, Philippine and most EBN products were consumed in these areas as well as in North America. Trade scale of global market has been going up for decades. From 1989 to 2004 trade value rose from HK$1.3 billion to HK $3 billion in Hong Kong area (Chan, 2009). In order to improve the quantity and quality of EBN and to protect the natural environment of swiftlet residence, man-made bird house was built widely.
⁎ Corresponding author. Chinese Academy of Inspection and Quarantine, No.3, Gaobeidian North Street, Chaoyang District, Beijing, 100123, China. Fax: +86 10 85772995. E-mail address: [email protected] (Y. Chen). 0963-9969/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2010.05.020
However, the tension between supply and need exists because of low production level (about 2000 t per year). As a result, the number of documented and suspected cases of EBN adulteration with less expensive materials has risen sharply (Marcone, 2005). Various fake materials were added to increase the net weight of the nest prior to sale including Tremella fungus, karaya gum, red seaweed, pork skin and egg white, etc (Wu et al., 2007). These adulterants were usually incorporated at levels approximating 10% and were extremely difficult to detect due to their similar color, appearance, taste and texture to the actual salivary nest cement (Marcone, 2005). Scientific analysis of EBN ingredient could be traced back to (Wang, 1921). But so far no official method could be found for quality surveillance of EBN products, though a few literatures on EBN authentication were published ever since 1990 (Sam & Tan, 1991). Anatomical trait and chemical composition of EBN was examined. For example, EBN specific fiber array was observed through scanning electron microscopy (SEM) (Sam & Tan, 1991) or X-ray microanalysis (Marcone, 2005). However, SEM did not prove to be an effective technique except for those containing red seaweed (Marcone, 2005). Spectrophotometry method was established to evaluate the content of sialic acid (9%), a nine-carbon sugar, which is one of the major components in EBN (Huang & Chen, 2003). EBN saccharide, such as mannitol, galactose, NAcGal, NAcGlc, were determined by GC (Yu et al, 1998; Yu & Xue, 2000). Composition of amino acid was detected by CE-GC method (Chen, 1995) and content of protein, amino acid and polysaccharide was measured by FIRT method (Sun & Liang, 2001). Amounts of various nutritionally important minerals were analyzed by atomic absorption (Marcone, 2005). Although significant difference of chemical fingerprint was determined between EBN and other
Y. Wu et al. / Food Research International 43 (2010) 2020–2026 Table 1 Summary of 2DGE spot matching among EBN and other samples by means of PDQuest software. No
Origin
Sample name
Spot
Automatic match rate
Manual match rate
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Malaysia
SUPU* ROOF WHITE WHITE WHITE WHITE GUAMU GUAMAN GUANYAN JINSIYAN LONGQING DONGFANGHONG JINSIYAN JINSIYAN Tremella fungus Milk Soybean Rice
29 8 14 11 18 19 9 9 18 23 21 8 22 25 62 194 70 86
100% 66% 42% 55% 68% 38% 66% 64% 32% 43% 39% 73% 44% 43% 21% 5% 11% 10%
100% 86% 95% 100% 92% 96% 100% 85% 88% 70% 82% 86% 75% 84% 26% 38% 16% 11%
Indonesia
China Vietnam Thailand
Local market
i.*Master sample. ii. During automatic matching only protein size markers were matched manually and sample protein was matched to master sample automatically with matching precision parameter set as 50. iii. During manual matching apparently identical protein spots between master sample and member sample was matched. An example of manual matching was provided in Fig. 6B.
materials in these studies, little information on EBN from different geographical areas was provided. Besides, in vivo synthesis of polysaccharide was not as stringently modeled as DNA or protein and could easily be affected by change of micro-environment (Lin et al., 2006). As the most abundant component in EBN was crude protein (more than 60%) (Marcone, 2005), protein profiling based on SDSPAGE method was also conducted (Hu and Lai, 1999). For EBN authentication, various methods were developed for different regulatory purposes. Herein we reported a combination of PCR and 2DGE methods, where PCR was applied for rapid judgment of the presence of EBN and 2DGE was used to detect heterogenous protein from adulterant. The combination of pI and MW parameters dramatically enhance resolution power of 2DGE compared to SDS-PAGE and it became a cornerstone technique in proteome research. 2. Materials and methods 2.1. Sample preparation Fourteen EBN samples from different countries (as shown in Table 1) were supplied by Guang Dong CIQ and Hu Qin Yu Tang Health Product Company. Other biological materials including Tremella fungus, duck, chicken, pigeon, beef, pork, milk, soybean, rice, etc. were purchased from the local market. All solid samples were ground into particles and stored at 4 °C.
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2.2. DNA extraction and quantification Three methods were tried to extract DNA from EBN. CTAB method was conducted as reported by Corbisier (2007). Wizard® food DNA purification kit (Promega, Madison, USA) and Nucleospin® DNA purification kit (MACHEREY-NAGEL, Duren, Germany) were performed following the manuscript. DNA extracted from EBN was quantified by Nanodrop fluorescence spectrometry. 2.3. Preparation of water-soluble protein Water-soluble protein was extracted according to the method of Goh et al. (2001) with some modification. For each sample, 500 mg material was suspended in 10 ml distilled water and allowed to incubate for 24 h at 4 °C. Supernatant (about 3.6 ml) was collected and distributed in 6 clean microtubes (600 μl per tube) which was then centrifuged in a vacuum concentrator (Concentrator plus 5305, Eppendorf, Germany) until the liquid was reduced to 100 μl. The solution was desalted and condensed using ReadyPrep 2-D Cleanup kit (Biorad, Hercules, California, USA) following the instruction. During condensation, the protein solution was freeze-dried as required in the instruction. The resulting pellet was stored at −20 °C for 2DGE analysis. 2.4. SYBRgreen PCR PCR reaction was performed in 25 μl system containing 1 × SYBRgreen Master mix (Roche, Indianapolis, USA) and 5 μl DNA template. Swiftlet beta-fibrinogen gene was amplified (161 bp). The primer pair was designed as following: cfib-F: 5′-TGAATTGAGCCTGTCGTCTG-3′ cfib-R: 5′-TGCTCCATAGCCAAACAAGA-3′ Thermo parameters were set as 95 °C 15 min for initial denaturing, 95 °C 20 s and 60 °C 1 min for 45 cycles, 95 °C 15 s, 60 °C 20 s and 95 °C 15 s for melting temperature measurement. Ramp time between the last two hold was set as 19 min and 59 s. Reaction was performed on IQ5 (Biorad, Hercules, California, USA). 2.5. 2DGE of proteins Lyophilized protein sediment was resolved in rehydration buffer (ReadyStrip pH4.7–5.9 buffer: pH6.3–8.3 buffer = 2:1 v/v) with the help of ultrasonic treatment (Uibracell VCX750, Sonics and Materials Inc, USA). The sample was used to hydrate 11 cm pH4.7–5.9 IPG strip for 12 h at room temperature. Hydrated IPG strip was focused in a PROTEAN IEF System (Biorad, Hercules, California, USA) at 250 V for 30 min followed by 1000 V for 30 min before the voltage was increased to 8 kV for a total of 40 kVh. In the second dimensional SDS-PAGE assay, focused strips were first balanced in equilibrium buffer I and buffer II (Biorad, Hercules, California, USA), then embedded with 0.5% agarose on top of 10% polyacrylamide gels (13.3 × 8.7 cm). Electrophoresis was performed in Criterion Cell (Biorad, Hercules, California, USA) at 5 mA/gel for 1 h followed by 20 mA/gel for 3 h. Gel was stained with Coomassie Brilliant Blue G250 and destained in 1% acetic acid. Images were captured on
Fig. 1. Multi-allignment of E1 beta-fibrinogen target sequence among Collocalia and other genera using online ClustalW2 tool. Primer regions were covered by horizontal arrows and SNPs between Collocalia and the other genera were signified by vertical arrows.
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Versadoc Imager (Biorad, Hercules, California, USA) in transmission mode. 3. Results and discussion Because counterfeit EBN food often contained no EBN component, we first developed an EBN specific DNA method to rapidly discover entirely fraud product. Primers were designed based on E1 betafibrinogen gene sequence (AY513092) using online Primer-BLAST tool. Primer efficiency was investigated and the primer pair with highest sensitivity was selected. Blast of EBN beta-fibrinogen gene sequence against nr database revealed some genera phylogenetically
Fig. 2. Agarose gel electrophoresis result of beta-fibrinogen PCR product amplified from EBN DNA which was extracted with different methods (A) and SYBRgreen melting curves of PCR product (B). DNA extracted from 200 mg, 100 mg, 50 mg, 20 mg and 10 mg EBN using Nucleospin kit method was quantified by Nanodrop fluorescence spectrometry and correlation between DNA concentration and sample size was plotted (C). M: DNA ladder (up to down 600 500 400 300 200 100 bp); 1. Blank control; 2, 3. CTAB method 4 5.Promega Wizard kit 6, 7. Nucleospin kit 8,9. Chicken DNA; 10, 11. Duck DNA. PCR product of EBN DNA was expected to be 161 bp and electrophoresis result showed a single and strong band close to 200 bp. The melting temperature of PCR product of EBN was shown as 76.05 °C, distinct from chicken (78 °C) and duck (79 °C). In the assay of DNA quantification, three independent extractions were performed for each sample size and for each extraction DNA concentration was determined according to the mean value of triple reading of the spectrometer. The quantification standard curve was formed automatically by the instrument.
close to Collocalia with similar beta-fibrinogen gene sequence. As shown in Fig. 1, multi-alignment of beta-fibrinogen target sequence among two Collocalia species and 7 other close genera demonstrated that two Collocalia species were identical and SNPs existed between Collocalia and the other genera, implying the possibility of taking advantage of the sequence heterogeneity to differentiate EBN and the other species. In this study, SYBRgreen PCR method was selected and EBN DNA was supposed to be identified in terms of specific melting temperature. Because EBN essentially is dried saliva mucus containing abundant glycoprotein and the end product usually undergoes heat treatment after harvest, it is difficult to recover efficient and qualified DNA from EBN. In our study the routine CTAB method, Wizard® food DNA purification kit method and Nucleospin® DNA purification kit method were compared and DNA quality was evaluated through PCR. As shown in Fig. 2A and B, EBN DNA extracted by the three methods (lanes 2–7) as well as chicken and duck DNA (lanes 8–11) used as negative control was amplified. Within the three methods, only DNA recovered by Nucleospin kit method was successfully amplified. Agarose gel electrophoresis results showed a single and strong band close to 200 bp which was supposed to be the target product since the expected amplicon was 161 bp. Although Nucleospin method was found to be more efficient for EBN than the other two methods, there was report about Wizard food DNA purification kit performing better than Nucleospin kit for tomato derived food (Manuela & Marmiroli, 2009). Therefore selection of appropriate method for DNA extraction should depend on experimental result on particular food type. In order to substantiate the efficiency of Nucleospin method, different
Fig. 3. Correlation plots between Ct value and logarithm of pure EBN DNA concentration (A) and between Ct value and logarithm of EBN weight percentage in EBN/ Tremella fungus mixture (B). Ct value of SYBRgreen PCR was recorded automatically by the machine. Pure EBN DNA was five fold diluted from 800 ng/ml to 0.16 ng/ml. EBN powder was mixed with Tremella fungus powder at 100%, 50%, 10%, 5%, and 0.5%. For each sample triple PCR reactions were conducted and mean Ct value was calculated.
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amount of EBN powder was treated simultaneously and recovered DNA was examined using fluorospectrometer (ND3300, Nanodrop Inc., USA). Based on the standard quantification curve of reference DNA provided in the SYBRgreenI DNA quantification kit, gross quantity of DNA recovered from EBN was decided. As shown in Fig. 2C, positive linear correlation between DNA quantity and sample size (R2 = 0.98) indicated good response of the method to sample size. Additionally, electrophoresis of PCR product of chicken and duck DNA did not show any visible band on the gel. However, SYBRgreen PCR melting curve of these two species revealed weak signals of product peak and their Tm values (78 °C and 79 °C) were distinct from EBN (76 °C), proving that SYBRgreen PCR was more sensitive than routine PCR and EBN could be identified by its specific Tm value. To analyze the efficiency of the SYBRgreen PCR system in identifying EBN originated DNA, pure EBN DNA was diluted from 800 ng/ml to 0.16 ng/ml and logarithm of DNA concentration was plotted against mean Ct value of each DNA sample. Good linear correlation (Fig. 3A) (R2 = 0.99) confirmed high efficiency of the PCR system (N90%) and high purity of DNA template (no inhibitor). We further prepared EBN/Tremella fungus mixtures at 100%, 50%, 10%, 5% and 0.5% to test detection limit of the method (Tremella fungus gave negative signal in SG-PCR). Correlation between logarithm of EBN percentage and Ct value of each mixture was shown as Fig. 3B (R2 = 0.96). Detection limit was as low as 0.5% EBN for EBN/Tremella fungus mixtures. To further examine the consistency of the method in
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identifying EBN originated from different areas as well as the specificity in distinguishing EBN from other species, 10 EBN samples from 5 countries (Malaysia, Indonesia, China, Vietnam, and Thailand) and 20 other biological species were tested. Part of the melting curves was shown in Fig. 4. The assay proved that Tm values of PCR products of ten EBN samples originated from different countries were identical, while Tm value of EBN PCR product was distinct from those of all the other species. Moreover, each sample was assayed repeatedly for 2–4 times and the shape of melting curves was highly similar for all repeated reaction, indicating good reproducibility of the method. DNA based method is widely accepted as the most powerful tool in species identification. Nevertheless, DNA method usually targeted at specific species, thus particular primer or probes needs to be designed beforehand. For unknown material mixed in the product, it is hard to design primer or probe without specific information. Since nearly all biological materials possessed unique protein composition, protein profiling technique would be more versatile in detecting adulterant. Early in 1999 2DGE technique was applied in commercial fish species identification (Carmen et al., 1999). Later on a lot of research works were published in this field, such as milk (Miranda et al., 2004; Holland et al., 2004; Joss et al., 2007; Kuy et al., 2007; Caroline & Hogarth, 2004), meat (Lametsch et al., 2002; Remignon, 2005), legume (Carbonaro et al., 2000), seafood (Martinez, 2004; Pineiro et al., 1999). Overview articles were written on the application of proteomics technology in food quality evaluation (Carbonaro, 2004), meat
Fig. 4. SYBRgreen PCR melting curves of 10 EBN samples originated from different countries and 5 other biological materials. 1. 10 EBN samples from Malaysia (SUPU, ROOF, WHITE), Indonesia (WHITE, GUAMU, GUAMAN), China (GUANYAN, JINSIYAN), Vietnam (LONGQING), Thailand (JINSIYAN); 2. EBN and chicken; 3. EBN and duck; 4. EBN and pigeon; 5. EBN and beef; 6. EBN and pork. For each sample 2–4 replicate reactions were performed.
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science (Bendixen, 2005), food nutrition (Kussmann et al., 2005) and milk science (Manso et al., 2005), implying a widespread application of proteomics technique in food authentication. To our knowledge 2DGE was also applied in EBN allergen research (Goh et al. 2001), but so far no report on application of 2DGE in EBN authentication was found. During the optimization of 2DGE method, we compared various protein extraction methods and selected among dry strips of different pH ranges. As long as operation simplicity and map quality were considered, we finally selected water incubation method and EBN water-soluble protein was best separated on the pH4.9–5.7 gel with the ratio between pH4.9–5.7 ampholyte and pH6.3–8.3 ampholyte as 2:1. 2DGE diagrams (Gaussie format) of SUPU EBN (Malaysia, SUPU is the name of a cave where high quality EBN was produced) were presented in Fig. 5 with 800 mg initial sample size (A), 500 mg sample size (B) and 100 mg sample size (C) respectively. Good reproducibility of spot pattern among the three maps could be visualized. Software analysis of spot intensity showed that with the decrease of sample size the signal intensity of majority spots lowered down (as shown by the column graph in diagram A), indicating that commassie blue dying of 2DGE gel could to some degree reflects the quantity of protein recovered from sample. Therefore the ratio between genuine EBN and adulterant is likely to be reflected by the ratio between EBN protein intensity and adulterant protein intensity. However, since some proteins were less sensitive to the change of sample size due to reasons such as diverse responses of different proteins to commassie blue (McDonald, 2009) more work needs to be done to determine the dynamic range of signal intensity of EBN protein and adulterant protein. In order to understand the characteristics of EBN protein, 14 EBN samples from different countries (1–4 Malyasia, 5–8 Indonesia, 9–10 China, 11 Vietnam, and 12–14 Thailand) were examined by 2DGE. As
shown in Fig. 6A, similar pattern was found for these geographical samples characterized by protein size between 57 kb and 28 kb, major protein pI in the middle between 4.7 and 5.9 and 1–4 lines of protein isomers clustered together in horizontal strings with identical molecular size but slightly different pI. These features were also found in 2D profile reported by Goh. However compared to 2D map of fresh EBN (Goh et al. 2001), our reference samples comprised much less amount of protein. The reason may be that during the afterharvesting processing fresh nest was cleaned by soaking it in water for many hours and afterwards it was remolded and air-dried, which may lead to protein loss and degradation. Based on the unique 2D pattern of EBN proteome, it is possible to detect adulteration of other cheap materials. The difference between EBN and Tremella fungus (Fig. 6A15) was significant in terms of spot amount, molecular size and pI value of overall protein pattern. When they were mixed together both EBN and Tremella fungus proteins were co-presented in the gel with their own MW and pI features. As shown in Fig. 6A16, EBN specific protein was revealed (marked by oval) as mentioned above and Tremella fungus originated protein was found in other regions. The relative simpler and special pattern of EBN protein make the detection of adulterant protein easier, which means fraud material could be detected through matching between unknown sample and reference EBN sample. In order to improve the accuracy in map matching, software analysis should be applied in addition to visual observe. As shown in Fig. 6B, due to slight discrepancy of protein position among gels, manual matching was necessary to match proteins with similar MW and pI parameters. A summary of 2DGE spot matching result among EBN and other samples was presented in Table 1. For the 14 EBN samples originated from different countries, the total number of protein spot for each sample was ranged from 8 to 29. As compared to EBN, much more protein was recovered from the
Fig. 5. 2DGE profiles of EBN (SUPU, Malaysia) in three independent assays with different sample sizes as 800 mg (A), 500 mg (B) and 200 mg (C) respectively. Normalized comparison of dye intensity among three gels was conducted using PDQuest software and the signal intensity of some spots was shown as column graph in FigA. The spot ID was generated automatically by the software as shown by the graph and the average intensity of spots was as the following: ssp2508-62328 INT area, ssp2001-35436 INT area, ssp330129043 INT area, ssp4301-34802 INT area, ssp5501-29165 INT area, ssp6401-10233 INT area, ssp6301-24551 INT area, ssp5301-79964 INT area.
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other materials (Tremella fungus, milk, soybean and rice). When all the 2D patterns were analyzed without manual assistance (automatic match), it showed that match rate of EBN samples as relative to SUPU EBN (the designated master sample) ranged from 32% to 73%, while the rate of the other materials from 5% to 21%. However, when manual match was performed before calculation, match rate of EBN was improved to 70%–100% and the rate of the other material 11%–38%. Therefore through manual assistance genuine EBN could easily be differentiated from other materials (match rate ≥ 70%) and the decrease of match rate would imply the existence of fraud material. To examine the accuracy and detection limit of the method, five groups of 100%, 90%, 50% EBN/Tremella fungus and 100% Tremella fungus were tested and their protein profiles were analyzed by PDQuest under manual help as mentioned before. For each group, EBN from different countries was used. The match rates of each sample as relative to 100% EBN were shown in Fig. 7. For all five groups, match rate decreased when the ratio of fungus increased. The result demonstrated that as low as 10% Tremella fungus could be detected. In present work, only Tremella fungus was compared as a kind of
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regularly used adulterant. In the next work other frequently used adulterant will be included. In this study, both DNA and protein based methods were explored to identify genuine EBN and adulterant. SYBRgreen PCR system targeting at fibrinogen gene proved to be able to detect swiftlet ingredient specifically and sensitively. The method could be used in rapid diagnosis of complete fake product, which is not unusual in market. At the same time it could be applied for sensitive detection of EBN ingredient as a potential allergen. 2DGE method analyzes EBN originated water-soluble proteins by pI and MW parameters. 2D profiles of fourteen EBN samples from different countries revealed shared feature which was useful in distinguishing EBN and adulterant. Software analysis of 2D profile demonstrated that under the assistance of manual match the match rate of EBN samples relative to SUPU EBN was much higher than the other materials and as low as 10% Tremella fungus could be detected from EBN/Tremella fungus mixture. In present work, only four samples including Tremella fungus, rice, soybean and milk were compared with EBN by 2DGE method. With
Fig. 6. 2DGE profiles of 14 EBN samples originated from different countries as well as Tremella fungus (A) and a presentation of manual match between profile1 and profile6 (B). M indicates protein molecular weight standards (194 kD 104 kD 57 kD 41 kD 28 kD 21 kD and 16 kD from up to down). The number of profiles 1–15 was corresponding to the sample number of Table 1and profile 16 was 50% EBN/Tremella fungus mixture in which EBN derived proteins were emphasized by oval. An example of manual match was shown in part B. Protein spots with identical alphabet were matched.
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Fig. 7. Comparison of match rate of 5 groups of EBN/Tremella fungus mixtures (100% EBN, 90%EBN, 50%EBN, 100%fungus). For each group EBN from different countries was used. 1. Malaysia SUPU; 2. Indonesia GUAMU; 3. China GUANYAN; 4. Vietnam LONGQING; 5. Thailand JINSIYAN.
the establishment of 2DGE database in our laboratory, other adulterant will be included. As for karaya gum which is a dried exudation of the stem and branches of Sterculia urens, its protein content (0.7% reported by Marcone, 2005) was so low that no protein was found on 2D map (result not shown). Therefore we supposed that for pigment or polysaccharides products the appropriate tool is PCR targeting at specific adulterants. Acknowledgement This work is supported by the Eleventh Five-year Plan project of the Ministry of Science and Technology, People's Republic of China (contract No:2006BAD27B02). We thank Dr. Huang Hua-jun from Guang Dong CIQ and Mr. Zhang Ji-tuo from Hu Qin Yu Tang Health Product Company for their generous supply of EBN samples. References Bendixen, E. (2005). The use of proteomics in meat science. Meat Science, 71, 138−149. Carbonaro, M., et al. (2000). Perspectives into factors limiting in vivo digestion of legume proteins: antinutritional compounds or storage proteins. Journal of Agricultural and Food Chemistry, 48(3), 742−749. Carbonaro, M. (2004). Proteomics: Present and future in food quality evaluation. Trends in Food Science and Technology, 209−216. Carmen, P., et al. (1999). Development of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis reference method for the analysis and identification of fish species
in raw and heat-processed samples: A collaborative study. Electrophoresis, 20(7), 1425−1432. Caroline, J., & Hogarth, J. L. (2004). Differential protein composition of bovine whey: A comparison of whey from healthy animals and from those with clinical mastitis. Proteomics, 2094−2100. Chan, S. W. (2009). Review of scientific research on edible bird's nest. http://ww. hkfsta.com.hk/articles/special/article7.htm. Chen, W. R. (1995). Determination of amino acid and authentication of EBN by CE-GC method. Chinese journal of tour medical science, 1, 110−114. Corbisier, P. B. (2007). Toward metrological traceability for DNA fragment ratios in GM quantification. Journal of Agriculture and Food Chemistry, 55, 3249−3257. Goh, D. L. M., et al. (2001). Immunochemical characterization of edible bird's nest allergens. Journal of Allergy Clinical Immunology, 107, 1082−1088. Holland, J. W., et al. (2004). Proteomic analysis of k-casein micro-heterogeneity. Proteomics(4), 743−752. Hu, S. M., & Lai, D. M. (1999). SDS-PAGE idenfication of edible bird's nest. Journal of Chinese medicine, 24, 331−334. Huang, H., & Chen, X. X. (2003). Determination of content of bird nest by spectrophotometer. Guangzhou Food Science and Technology, 19, 68−71. Joss, J., et al. (2007). Proteomic analysis of early lactation milk of the tammar wallaby (Macropus eugenii). Comparative Biochemistry and Physiology(D2), 150−164. Kussmann, M., et al. (2005). Combinatorial chemistry and high throughput screening. Proteomics in Nutrition and Health(8), 679−696. Kuy, S., et al. (2007). Proteomic analysis of whey and casein proteins in early milk from the marsupial Trichosurus vulpecula, the common brushtail possum. Comparative Biochemistry and Physiology(D2), 112−120. Lametsch, R., et al. (2002). Identification of protein degradation during post-mortem storage of pig meat. Journal of Agricultural and food chemistry, 5508−5512. Lin, J., et al. (2006). Overview of edible bird's nest research. Chinese medicine, 29, 58−64. Manso, M. A., et al. (2005). Application of proteomics to the characterization of milk and dairy products. International dairy journal(15), 845−855. Manuela, T. M., & Marmiroli, N. (2009). Evaluation of DNA extraction procedures for traceability of various tomato products. Food control, 21, 143−149. Marcone, M. F. (2005). Characterization of the edible bird's nest the “Caviar of the East”. Food research international, 38, 1125−1134. Martinez, I. (2004). Application of proteome analysis to seafood authentication. Proteomics, 4, 347−354. McDonald, K. (2009). Overcoming the Coomassie Blues. Drug discovery & development. http://www.dddmag.com/Article-OvercomingtheCoomas-SieBlues-060409.aspx Miranda, G., et al. (2004). Proteomic tools to characterize the protein fraction of Equidae milk. Proteomics(4), 2496−2509. Pineiro, C., et al. (1999). The use of two-dimensional electrophoresis in the characterization of the water-soluble protein fraction of commercial flat fish species. Z Lebensm Unters Forsch A(208), 342−348. Remignon, H. (2005). Current advances in proteomic analysis and its use for the resolution of poultry meat quality problems. The XVIIth European Symposium on the Quality of Poultry Meat, Dooorwerth, The Netherlands. Sam, C. T., & Tan, P. H. L. (1991). Establishing the authenticity of edible bird's nest. ISFM medicine scientific review. Sun, S. Q., & Liang, X. Y. (2001). FIRT method applied for rapid identification of 6 edible bird's nest. Analytical chemistry, 29, 552−554. Wang, C. C. (1921). The composition of Chinese edible birds' nests and the nature of their proteins. Journal of Biological Chemistry, 429−439. Wu, R. H., et al. (2007). Review of EBN authentication method. Inspection and quarantine science, 17, 61−62. Yu, Y. Q., & Xue, L. (2000). Determination of edible bird's nest and its products by gas chromatography. Journal of Chromatographic Science, 38, 27−32. Yu, Y. Q., et al. (1998). Identification and quantication of Edible bird's nest product by GC. Journal of Instrumential Analysis, 17, 33−37.
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Application of SYBRgreen PCR and 2DGE methods to authenticate edible bird's nest food Yajun Wu a, Ying Chen a,⁎, Bin Wang a, Liqun Bai a, Wu ri han a, Yiqiang Ge b, Fei Yuan a a b
Chinese Academy of Inspection and Quarantine, No.3, Gaobeidian North Street, Chaoyang District, Beijing, China College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China
a r t i c l e
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Article history: Received 31 March 2010 Accepted 30 May 2010 Keywords: Edible bird's nest Authentication SYBRgreen PCR 2DGE
a b s t r a c t Edible bird's nest (EBN) as a special kind of food tonic has been highly esteemed in Chinese cuisine and medicinal culture. Particularly with the discovery of its healthy function by modern science, consumption of EBN food gained greater popularity within Chinese community and outside. Authentication of this precious and expensive food material became an urgent task facing the increasing occurrence of adulteration in the market. Herein we reported the combination of DNA based PCR and protein based two dimensional gel electrophoresis (2DGE) methods for rapid and reliable identification of genuine EBN product. Fourteen EBN samples from different countries were studied. PCR method was proved to be able to differentiate EBN and the other biological materials and it could detect EBN ingredient from 0.5% EBN/Tremella fungus mixture. 2DGE method was proved to be feasible and versatile in EBN identification because of the simple and unique protein pattern of EBN. The method could detect 10% Tremella fungus from EBN. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction EBN originated from the saliva of four swiftlet species: Collocalia fuciphaga, C. germanis, C. maxima and C. unicolor (Goh et al., 2001). Due to the highly evaluated function both nutritiously (water-soluble protein, carbohydrate, iron, inorganic salt and fiber) and medically (anti-aging, anti-cancer, immunity-enhancing, etc), EBN has been esteemed a precious food tonic by Chinese people ever since the Tang dynasty (618 AD). It was even referred as “Caviar of the East”. Current scientific study confirmed that EBN has haemagglutination inhibiting activity against the influenza virus and contained avian epidermal growth factor (Marcone, 2005; Lin et al., 2006). Nowadays with the help of modern commercialization and technology, EBN was developed into various food products including drink, food additive, and even cosmetic ingredient. At present EBN raw material mainly comes from Asian countries, such as China, Malaysia, Indonesia, Vietnam, Thailand, Philippine and most EBN products were consumed in these areas as well as in North America. Trade scale of global market has been going up for decades. From 1989 to 2004 trade value rose from HK$1.3 billion to HK $3 billion in Hong Kong area (Chan, 2009). In order to improve the quantity and quality of EBN and to protect the natural environment of swiftlet residence, man-made bird house was built widely.
⁎ Corresponding author. Chinese Academy of Inspection and Quarantine, No.3, Gaobeidian North Street, Chaoyang District, Beijing, 100123, China. Fax: +86 10 85772995. E-mail address: [email protected] (Y. Chen). 0963-9969/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2010.05.020
However, the tension between supply and need exists because of low production level (about 2000 t per year). As a result, the number of documented and suspected cases of EBN adulteration with less expensive materials has risen sharply (Marcone, 2005). Various fake materials were added to increase the net weight of the nest prior to sale including Tremella fungus, karaya gum, red seaweed, pork skin and egg white, etc (Wu et al., 2007). These adulterants were usually incorporated at levels approximating 10% and were extremely difficult to detect due to their similar color, appearance, taste and texture to the actual salivary nest cement (Marcone, 2005). Scientific analysis of EBN ingredient could be traced back to (Wang, 1921). But so far no official method could be found for quality surveillance of EBN products, though a few literatures on EBN authentication were published ever since 1990 (Sam & Tan, 1991). Anatomical trait and chemical composition of EBN was examined. For example, EBN specific fiber array was observed through scanning electron microscopy (SEM) (Sam & Tan, 1991) or X-ray microanalysis (Marcone, 2005). However, SEM did not prove to be an effective technique except for those containing red seaweed (Marcone, 2005). Spectrophotometry method was established to evaluate the content of sialic acid (9%), a nine-carbon sugar, which is one of the major components in EBN (Huang & Chen, 2003). EBN saccharide, such as mannitol, galactose, NAcGal, NAcGlc, were determined by GC (Yu et al, 1998; Yu & Xue, 2000). Composition of amino acid was detected by CE-GC method (Chen, 1995) and content of protein, amino acid and polysaccharide was measured by FIRT method (Sun & Liang, 2001). Amounts of various nutritionally important minerals were analyzed by atomic absorption (Marcone, 2005). Although significant difference of chemical fingerprint was determined between EBN and other
Y. Wu et al. / Food Research International 43 (2010) 2020–2026 Table 1 Summary of 2DGE spot matching among EBN and other samples by means of PDQuest software. No
Origin
Sample name
Spot
Automatic match rate
Manual match rate
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Malaysia
SUPU* ROOF WHITE WHITE WHITE WHITE GUAMU GUAMAN GUANYAN JINSIYAN LONGQING DONGFANGHONG JINSIYAN JINSIYAN Tremella fungus Milk Soybean Rice
29 8 14 11 18 19 9 9 18 23 21 8 22 25 62 194 70 86
100% 66% 42% 55% 68% 38% 66% 64% 32% 43% 39% 73% 44% 43% 21% 5% 11% 10%
100% 86% 95% 100% 92% 96% 100% 85% 88% 70% 82% 86% 75% 84% 26% 38% 16% 11%
Indonesia
China Vietnam Thailand
Local market
i.*Master sample. ii. During automatic matching only protein size markers were matched manually and sample protein was matched to master sample automatically with matching precision parameter set as 50. iii. During manual matching apparently identical protein spots between master sample and member sample was matched. An example of manual matching was provided in Fig. 6B.
materials in these studies, little information on EBN from different geographical areas was provided. Besides, in vivo synthesis of polysaccharide was not as stringently modeled as DNA or protein and could easily be affected by change of micro-environment (Lin et al., 2006). As the most abundant component in EBN was crude protein (more than 60%) (Marcone, 2005), protein profiling based on SDSPAGE method was also conducted (Hu and Lai, 1999). For EBN authentication, various methods were developed for different regulatory purposes. Herein we reported a combination of PCR and 2DGE methods, where PCR was applied for rapid judgment of the presence of EBN and 2DGE was used to detect heterogenous protein from adulterant. The combination of pI and MW parameters dramatically enhance resolution power of 2DGE compared to SDS-PAGE and it became a cornerstone technique in proteome research. 2. Materials and methods 2.1. Sample preparation Fourteen EBN samples from different countries (as shown in Table 1) were supplied by Guang Dong CIQ and Hu Qin Yu Tang Health Product Company. Other biological materials including Tremella fungus, duck, chicken, pigeon, beef, pork, milk, soybean, rice, etc. were purchased from the local market. All solid samples were ground into particles and stored at 4 °C.
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2.2. DNA extraction and quantification Three methods were tried to extract DNA from EBN. CTAB method was conducted as reported by Corbisier (2007). Wizard® food DNA purification kit (Promega, Madison, USA) and Nucleospin® DNA purification kit (MACHEREY-NAGEL, Duren, Germany) were performed following the manuscript. DNA extracted from EBN was quantified by Nanodrop fluorescence spectrometry. 2.3. Preparation of water-soluble protein Water-soluble protein was extracted according to the method of Goh et al. (2001) with some modification. For each sample, 500 mg material was suspended in 10 ml distilled water and allowed to incubate for 24 h at 4 °C. Supernatant (about 3.6 ml) was collected and distributed in 6 clean microtubes (600 μl per tube) which was then centrifuged in a vacuum concentrator (Concentrator plus 5305, Eppendorf, Germany) until the liquid was reduced to 100 μl. The solution was desalted and condensed using ReadyPrep 2-D Cleanup kit (Biorad, Hercules, California, USA) following the instruction. During condensation, the protein solution was freeze-dried as required in the instruction. The resulting pellet was stored at −20 °C for 2DGE analysis. 2.4. SYBRgreen PCR PCR reaction was performed in 25 μl system containing 1 × SYBRgreen Master mix (Roche, Indianapolis, USA) and 5 μl DNA template. Swiftlet beta-fibrinogen gene was amplified (161 bp). The primer pair was designed as following: cfib-F: 5′-TGAATTGAGCCTGTCGTCTG-3′ cfib-R: 5′-TGCTCCATAGCCAAACAAGA-3′ Thermo parameters were set as 95 °C 15 min for initial denaturing, 95 °C 20 s and 60 °C 1 min for 45 cycles, 95 °C 15 s, 60 °C 20 s and 95 °C 15 s for melting temperature measurement. Ramp time between the last two hold was set as 19 min and 59 s. Reaction was performed on IQ5 (Biorad, Hercules, California, USA). 2.5. 2DGE of proteins Lyophilized protein sediment was resolved in rehydration buffer (ReadyStrip pH4.7–5.9 buffer: pH6.3–8.3 buffer = 2:1 v/v) with the help of ultrasonic treatment (Uibracell VCX750, Sonics and Materials Inc, USA). The sample was used to hydrate 11 cm pH4.7–5.9 IPG strip for 12 h at room temperature. Hydrated IPG strip was focused in a PROTEAN IEF System (Biorad, Hercules, California, USA) at 250 V for 30 min followed by 1000 V for 30 min before the voltage was increased to 8 kV for a total of 40 kVh. In the second dimensional SDS-PAGE assay, focused strips were first balanced in equilibrium buffer I and buffer II (Biorad, Hercules, California, USA), then embedded with 0.5% agarose on top of 10% polyacrylamide gels (13.3 × 8.7 cm). Electrophoresis was performed in Criterion Cell (Biorad, Hercules, California, USA) at 5 mA/gel for 1 h followed by 20 mA/gel for 3 h. Gel was stained with Coomassie Brilliant Blue G250 and destained in 1% acetic acid. Images were captured on
Fig. 1. Multi-allignment of E1 beta-fibrinogen target sequence among Collocalia and other genera using online ClustalW2 tool. Primer regions were covered by horizontal arrows and SNPs between Collocalia and the other genera were signified by vertical arrows.
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Versadoc Imager (Biorad, Hercules, California, USA) in transmission mode. 3. Results and discussion Because counterfeit EBN food often contained no EBN component, we first developed an EBN specific DNA method to rapidly discover entirely fraud product. Primers were designed based on E1 betafibrinogen gene sequence (AY513092) using online Primer-BLAST tool. Primer efficiency was investigated and the primer pair with highest sensitivity was selected. Blast of EBN beta-fibrinogen gene sequence against nr database revealed some genera phylogenetically
Fig. 2. Agarose gel electrophoresis result of beta-fibrinogen PCR product amplified from EBN DNA which was extracted with different methods (A) and SYBRgreen melting curves of PCR product (B). DNA extracted from 200 mg, 100 mg, 50 mg, 20 mg and 10 mg EBN using Nucleospin kit method was quantified by Nanodrop fluorescence spectrometry and correlation between DNA concentration and sample size was plotted (C). M: DNA ladder (up to down 600 500 400 300 200 100 bp); 1. Blank control; 2, 3. CTAB method 4 5.Promega Wizard kit 6, 7. Nucleospin kit 8,9. Chicken DNA; 10, 11. Duck DNA. PCR product of EBN DNA was expected to be 161 bp and electrophoresis result showed a single and strong band close to 200 bp. The melting temperature of PCR product of EBN was shown as 76.05 °C, distinct from chicken (78 °C) and duck (79 °C). In the assay of DNA quantification, three independent extractions were performed for each sample size and for each extraction DNA concentration was determined according to the mean value of triple reading of the spectrometer. The quantification standard curve was formed automatically by the instrument.
close to Collocalia with similar beta-fibrinogen gene sequence. As shown in Fig. 1, multi-alignment of beta-fibrinogen target sequence among two Collocalia species and 7 other close genera demonstrated that two Collocalia species were identical and SNPs existed between Collocalia and the other genera, implying the possibility of taking advantage of the sequence heterogeneity to differentiate EBN and the other species. In this study, SYBRgreen PCR method was selected and EBN DNA was supposed to be identified in terms of specific melting temperature. Because EBN essentially is dried saliva mucus containing abundant glycoprotein and the end product usually undergoes heat treatment after harvest, it is difficult to recover efficient and qualified DNA from EBN. In our study the routine CTAB method, Wizard® food DNA purification kit method and Nucleospin® DNA purification kit method were compared and DNA quality was evaluated through PCR. As shown in Fig. 2A and B, EBN DNA extracted by the three methods (lanes 2–7) as well as chicken and duck DNA (lanes 8–11) used as negative control was amplified. Within the three methods, only DNA recovered by Nucleospin kit method was successfully amplified. Agarose gel electrophoresis results showed a single and strong band close to 200 bp which was supposed to be the target product since the expected amplicon was 161 bp. Although Nucleospin method was found to be more efficient for EBN than the other two methods, there was report about Wizard food DNA purification kit performing better than Nucleospin kit for tomato derived food (Manuela & Marmiroli, 2009). Therefore selection of appropriate method for DNA extraction should depend on experimental result on particular food type. In order to substantiate the efficiency of Nucleospin method, different
Fig. 3. Correlation plots between Ct value and logarithm of pure EBN DNA concentration (A) and between Ct value and logarithm of EBN weight percentage in EBN/ Tremella fungus mixture (B). Ct value of SYBRgreen PCR was recorded automatically by the machine. Pure EBN DNA was five fold diluted from 800 ng/ml to 0.16 ng/ml. EBN powder was mixed with Tremella fungus powder at 100%, 50%, 10%, 5%, and 0.5%. For each sample triple PCR reactions were conducted and mean Ct value was calculated.
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amount of EBN powder was treated simultaneously and recovered DNA was examined using fluorospectrometer (ND3300, Nanodrop Inc., USA). Based on the standard quantification curve of reference DNA provided in the SYBRgreenI DNA quantification kit, gross quantity of DNA recovered from EBN was decided. As shown in Fig. 2C, positive linear correlation between DNA quantity and sample size (R2 = 0.98) indicated good response of the method to sample size. Additionally, electrophoresis of PCR product of chicken and duck DNA did not show any visible band on the gel. However, SYBRgreen PCR melting curve of these two species revealed weak signals of product peak and their Tm values (78 °C and 79 °C) were distinct from EBN (76 °C), proving that SYBRgreen PCR was more sensitive than routine PCR and EBN could be identified by its specific Tm value. To analyze the efficiency of the SYBRgreen PCR system in identifying EBN originated DNA, pure EBN DNA was diluted from 800 ng/ml to 0.16 ng/ml and logarithm of DNA concentration was plotted against mean Ct value of each DNA sample. Good linear correlation (Fig. 3A) (R2 = 0.99) confirmed high efficiency of the PCR system (N90%) and high purity of DNA template (no inhibitor). We further prepared EBN/Tremella fungus mixtures at 100%, 50%, 10%, 5% and 0.5% to test detection limit of the method (Tremella fungus gave negative signal in SG-PCR). Correlation between logarithm of EBN percentage and Ct value of each mixture was shown as Fig. 3B (R2 = 0.96). Detection limit was as low as 0.5% EBN for EBN/Tremella fungus mixtures. To further examine the consistency of the method in
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identifying EBN originated from different areas as well as the specificity in distinguishing EBN from other species, 10 EBN samples from 5 countries (Malaysia, Indonesia, China, Vietnam, and Thailand) and 20 other biological species were tested. Part of the melting curves was shown in Fig. 4. The assay proved that Tm values of PCR products of ten EBN samples originated from different countries were identical, while Tm value of EBN PCR product was distinct from those of all the other species. Moreover, each sample was assayed repeatedly for 2–4 times and the shape of melting curves was highly similar for all repeated reaction, indicating good reproducibility of the method. DNA based method is widely accepted as the most powerful tool in species identification. Nevertheless, DNA method usually targeted at specific species, thus particular primer or probes needs to be designed beforehand. For unknown material mixed in the product, it is hard to design primer or probe without specific information. Since nearly all biological materials possessed unique protein composition, protein profiling technique would be more versatile in detecting adulterant. Early in 1999 2DGE technique was applied in commercial fish species identification (Carmen et al., 1999). Later on a lot of research works were published in this field, such as milk (Miranda et al., 2004; Holland et al., 2004; Joss et al., 2007; Kuy et al., 2007; Caroline & Hogarth, 2004), meat (Lametsch et al., 2002; Remignon, 2005), legume (Carbonaro et al., 2000), seafood (Martinez, 2004; Pineiro et al., 1999). Overview articles were written on the application of proteomics technology in food quality evaluation (Carbonaro, 2004), meat
Fig. 4. SYBRgreen PCR melting curves of 10 EBN samples originated from different countries and 5 other biological materials. 1. 10 EBN samples from Malaysia (SUPU, ROOF, WHITE), Indonesia (WHITE, GUAMU, GUAMAN), China (GUANYAN, JINSIYAN), Vietnam (LONGQING), Thailand (JINSIYAN); 2. EBN and chicken; 3. EBN and duck; 4. EBN and pigeon; 5. EBN and beef; 6. EBN and pork. For each sample 2–4 replicate reactions were performed.
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science (Bendixen, 2005), food nutrition (Kussmann et al., 2005) and milk science (Manso et al., 2005), implying a widespread application of proteomics technique in food authentication. To our knowledge 2DGE was also applied in EBN allergen research (Goh et al. 2001), but so far no report on application of 2DGE in EBN authentication was found. During the optimization of 2DGE method, we compared various protein extraction methods and selected among dry strips of different pH ranges. As long as operation simplicity and map quality were considered, we finally selected water incubation method and EBN water-soluble protein was best separated on the pH4.9–5.7 gel with the ratio between pH4.9–5.7 ampholyte and pH6.3–8.3 ampholyte as 2:1. 2DGE diagrams (Gaussie format) of SUPU EBN (Malaysia, SUPU is the name of a cave where high quality EBN was produced) were presented in Fig. 5 with 800 mg initial sample size (A), 500 mg sample size (B) and 100 mg sample size (C) respectively. Good reproducibility of spot pattern among the three maps could be visualized. Software analysis of spot intensity showed that with the decrease of sample size the signal intensity of majority spots lowered down (as shown by the column graph in diagram A), indicating that commassie blue dying of 2DGE gel could to some degree reflects the quantity of protein recovered from sample. Therefore the ratio between genuine EBN and adulterant is likely to be reflected by the ratio between EBN protein intensity and adulterant protein intensity. However, since some proteins were less sensitive to the change of sample size due to reasons such as diverse responses of different proteins to commassie blue (McDonald, 2009) more work needs to be done to determine the dynamic range of signal intensity of EBN protein and adulterant protein. In order to understand the characteristics of EBN protein, 14 EBN samples from different countries (1–4 Malyasia, 5–8 Indonesia, 9–10 China, 11 Vietnam, and 12–14 Thailand) were examined by 2DGE. As
shown in Fig. 6A, similar pattern was found for these geographical samples characterized by protein size between 57 kb and 28 kb, major protein pI in the middle between 4.7 and 5.9 and 1–4 lines of protein isomers clustered together in horizontal strings with identical molecular size but slightly different pI. These features were also found in 2D profile reported by Goh. However compared to 2D map of fresh EBN (Goh et al. 2001), our reference samples comprised much less amount of protein. The reason may be that during the afterharvesting processing fresh nest was cleaned by soaking it in water for many hours and afterwards it was remolded and air-dried, which may lead to protein loss and degradation. Based on the unique 2D pattern of EBN proteome, it is possible to detect adulteration of other cheap materials. The difference between EBN and Tremella fungus (Fig. 6A15) was significant in terms of spot amount, molecular size and pI value of overall protein pattern. When they were mixed together both EBN and Tremella fungus proteins were co-presented in the gel with their own MW and pI features. As shown in Fig. 6A16, EBN specific protein was revealed (marked by oval) as mentioned above and Tremella fungus originated protein was found in other regions. The relative simpler and special pattern of EBN protein make the detection of adulterant protein easier, which means fraud material could be detected through matching between unknown sample and reference EBN sample. In order to improve the accuracy in map matching, software analysis should be applied in addition to visual observe. As shown in Fig. 6B, due to slight discrepancy of protein position among gels, manual matching was necessary to match proteins with similar MW and pI parameters. A summary of 2DGE spot matching result among EBN and other samples was presented in Table 1. For the 14 EBN samples originated from different countries, the total number of protein spot for each sample was ranged from 8 to 29. As compared to EBN, much more protein was recovered from the
Fig. 5. 2DGE profiles of EBN (SUPU, Malaysia) in three independent assays with different sample sizes as 800 mg (A), 500 mg (B) and 200 mg (C) respectively. Normalized comparison of dye intensity among three gels was conducted using PDQuest software and the signal intensity of some spots was shown as column graph in FigA. The spot ID was generated automatically by the software as shown by the graph and the average intensity of spots was as the following: ssp2508-62328 INT area, ssp2001-35436 INT area, ssp330129043 INT area, ssp4301-34802 INT area, ssp5501-29165 INT area, ssp6401-10233 INT area, ssp6301-24551 INT area, ssp5301-79964 INT area.
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other materials (Tremella fungus, milk, soybean and rice). When all the 2D patterns were analyzed without manual assistance (automatic match), it showed that match rate of EBN samples as relative to SUPU EBN (the designated master sample) ranged from 32% to 73%, while the rate of the other materials from 5% to 21%. However, when manual match was performed before calculation, match rate of EBN was improved to 70%–100% and the rate of the other material 11%–38%. Therefore through manual assistance genuine EBN could easily be differentiated from other materials (match rate ≥ 70%) and the decrease of match rate would imply the existence of fraud material. To examine the accuracy and detection limit of the method, five groups of 100%, 90%, 50% EBN/Tremella fungus and 100% Tremella fungus were tested and their protein profiles were analyzed by PDQuest under manual help as mentioned before. For each group, EBN from different countries was used. The match rates of each sample as relative to 100% EBN were shown in Fig. 7. For all five groups, match rate decreased when the ratio of fungus increased. The result demonstrated that as low as 10% Tremella fungus could be detected. In present work, only Tremella fungus was compared as a kind of
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regularly used adulterant. In the next work other frequently used adulterant will be included. In this study, both DNA and protein based methods were explored to identify genuine EBN and adulterant. SYBRgreen PCR system targeting at fibrinogen gene proved to be able to detect swiftlet ingredient specifically and sensitively. The method could be used in rapid diagnosis of complete fake product, which is not unusual in market. At the same time it could be applied for sensitive detection of EBN ingredient as a potential allergen. 2DGE method analyzes EBN originated water-soluble proteins by pI and MW parameters. 2D profiles of fourteen EBN samples from different countries revealed shared feature which was useful in distinguishing EBN and adulterant. Software analysis of 2D profile demonstrated that under the assistance of manual match the match rate of EBN samples relative to SUPU EBN was much higher than the other materials and as low as 10% Tremella fungus could be detected from EBN/Tremella fungus mixture. In present work, only four samples including Tremella fungus, rice, soybean and milk were compared with EBN by 2DGE method. With
Fig. 6. 2DGE profiles of 14 EBN samples originated from different countries as well as Tremella fungus (A) and a presentation of manual match between profile1 and profile6 (B). M indicates protein molecular weight standards (194 kD 104 kD 57 kD 41 kD 28 kD 21 kD and 16 kD from up to down). The number of profiles 1–15 was corresponding to the sample number of Table 1and profile 16 was 50% EBN/Tremella fungus mixture in which EBN derived proteins were emphasized by oval. An example of manual match was shown in part B. Protein spots with identical alphabet were matched.
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Fig. 7. Comparison of match rate of 5 groups of EBN/Tremella fungus mixtures (100% EBN, 90%EBN, 50%EBN, 100%fungus). For each group EBN from different countries was used. 1. Malaysia SUPU; 2. Indonesia GUAMU; 3. China GUANYAN; 4. Vietnam LONGQING; 5. Thailand JINSIYAN.
the establishment of 2DGE database in our laboratory, other adulterant will be included. As for karaya gum which is a dried exudation of the stem and branches of Sterculia urens, its protein content (0.7% reported by Marcone, 2005) was so low that no protein was found on 2D map (result not shown). Therefore we supposed that for pigment or polysaccharides products the appropriate tool is PCR targeting at specific adulterants. Acknowledgement This work is supported by the Eleventh Five-year Plan project of the Ministry of Science and Technology, People's Republic of China (contract No:2006BAD27B02). We thank Dr. Huang Hua-jun from Guang Dong CIQ and Mr. Zhang Ji-tuo from Hu Qin Yu Tang Health Product Company for their generous supply of EBN samples. References Bendixen, E. (2005). The use of proteomics in meat science. Meat Science, 71, 138−149. Carbonaro, M., et al. (2000). Perspectives into factors limiting in vivo digestion of legume proteins: antinutritional compounds or storage proteins. Journal of Agricultural and Food Chemistry, 48(3), 742−749. Carbonaro, M. (2004). Proteomics: Present and future in food quality evaluation. Trends in Food Science and Technology, 209−216. Carmen, P., et al. (1999). Development of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis reference method for the analysis and identification of fish species
in raw and heat-processed samples: A collaborative study. Electrophoresis, 20(7), 1425−1432. Caroline, J., & Hogarth, J. L. (2004). Differential protein composition of bovine whey: A comparison of whey from healthy animals and from those with clinical mastitis. Proteomics, 2094−2100. Chan, S. W. (2009). Review of scientific research on edible bird's nest. http://ww. hkfsta.com.hk/articles/special/article7.htm. Chen, W. R. (1995). Determination of amino acid and authentication of EBN by CE-GC method. Chinese journal of tour medical science, 1, 110−114. Corbisier, P. B. (2007). Toward metrological traceability for DNA fragment ratios in GM quantification. Journal of Agriculture and Food Chemistry, 55, 3249−3257. Goh, D. L. M., et al. (2001). Immunochemical characterization of edible bird's nest allergens. Journal of Allergy Clinical Immunology, 107, 1082−1088. Holland, J. W., et al. (2004). Proteomic analysis of k-casein micro-heterogeneity. Proteomics(4), 743−752. Hu, S. M., & Lai, D. M. (1999). SDS-PAGE idenfication of edible bird's nest. Journal of Chinese medicine, 24, 331−334. Huang, H., & Chen, X. X. (2003). Determination of content of bird nest by spectrophotometer. Guangzhou Food Science and Technology, 19, 68−71. Joss, J., et al. (2007). Proteomic analysis of early lactation milk of the tammar wallaby (Macropus eugenii). Comparative Biochemistry and Physiology(D2), 150−164. Kussmann, M., et al. (2005). Combinatorial chemistry and high throughput screening. Proteomics in Nutrition and Health(8), 679−696. Kuy, S., et al. (2007). Proteomic analysis of whey and casein proteins in early milk from the marsupial Trichosurus vulpecula, the common brushtail possum. Comparative Biochemistry and Physiology(D2), 112−120. Lametsch, R., et al. (2002). Identification of protein degradation during post-mortem storage of pig meat. Journal of Agricultural and food chemistry, 5508−5512. Lin, J., et al. (2006). Overview of edible bird's nest research. Chinese medicine, 29, 58−64. Manso, M. A., et al. (2005). Application of proteomics to the characterization of milk and dairy products. International dairy journal(15), 845−855. Manuela, T. M., & Marmiroli, N. (2009). Evaluation of DNA extraction procedures for traceability of various tomato products. Food control, 21, 143−149. Marcone, M. F. (2005). Characterization of the edible bird's nest the “Caviar of the East”. Food research international, 38, 1125−1134. Martinez, I. (2004). Application of proteome analysis to seafood authentication. Proteomics, 4, 347−354. McDonald, K. (2009). Overcoming the Coomassie Blues. Drug discovery & development. http://www.dddmag.com/Article-OvercomingtheCoomas-SieBlues-060409.aspx Miranda, G., et al. (2004). Proteomic tools to characterize the protein fraction of Equidae milk. Proteomics(4), 2496−2509. Pineiro, C., et al. (1999). The use of two-dimensional electrophoresis in the characterization of the water-soluble protein fraction of commercial flat fish species. Z Lebensm Unters Forsch A(208), 342−348. Remignon, H. (2005). Current advances in proteomic analysis and its use for the resolution of poultry meat quality problems. The XVIIth European Symposium on the Quality of Poultry Meat, Dooorwerth, The Netherlands. Sam, C. T., & Tan, P. H. L. (1991). Establishing the authenticity of edible bird's nest. ISFM medicine scientific review. Sun, S. Q., & Liang, X. Y. (2001). FIRT method applied for rapid identification of 6 edible bird's nest. Analytical chemistry, 29, 552−554. Wang, C. C. (1921). The composition of Chinese edible birds' nests and the nature of their proteins. Journal of Biological Chemistry, 429−439. Wu, R. H., et al. (2007). Review of EBN authentication method. Inspection and quarantine science, 17, 61−62. Yu, Y. Q., & Xue, L. (2000). Determination of edible bird's nest and its products by gas chromatography. Journal of Chromatographic Science, 38, 27−32. Yu, Y. Q., et al. (1998). Identification and quantication of Edible bird's nest product by GC. Journal of Instrumential Analysis, 17, 33−37.